The present invention relates to glucose regulation of fatty acid synthesis and, more particularly, to a glucose-inducible human fatty acid synthase mRNA binding protein.
Long chain fatty acids are necessary for normal cellular structure and function. De novo synthesis represents an important source of these fatty acids. Critical to this process is the enzyme fatty acid synthase (FAS), a multifunctional protein which synthesizes saturated fatty acids (mostly palmitate) from acetyl-CoA, malonyl-CoA, and NADPH. FAS is believed to be involved in the pathogenesis of such diverse disorders as atherosclerosis and malignancies, FAS overexpression is associated with hyper-triglyceridemia, a common disorder predisposing to atherosclerosis. High levels of FAS predict a poor prognosis in humans with breast cancer.
(NOTE: Literature references on the following background information and on conventional test methods and laboratory procedures well-known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein, are indicated by reference numerals in parentheses, and appended at the end of the specification).
Chronically elevated concentrations of glucose like those seen in humans with diabetes mellitus are probably toxic. Insulin is important for glucose transport in certain tissues, but the majority of glucose uptake is independent of insulin and proportional to circulating glucose concentrations (6). In insulin-resistant states such as Type II diabetes, the total flux of glucose across certain tissues such as the liver is substantially increased. Glucose alone, independent of hormonal derangements, likely contributes to complications of diabetes such as hyperlipidemia (33).
Exactly how glucose promotes hyperlipidemia is unknown. The liver is central to this process and carbohydrates increase the expression of several hepatic genes involved in intermediary metabolism and lipogenesis including fatty acid synthase, L-type pyruvate kinase, acetyl-CoA carboxylase, ATP-citrate lyase, and malic enzyme (10). This effect is primarily transcriptional but glucose also stabilizes mRNAs for many feeding-responsive hepatic genes (32). Glucose promotes the stability of the message for the insulin receptor (12), another protein central to fuel flow and lipid metabolism.
Regulation of mRNA stability is an important mechanism for altering mammalian gene expression. Messenger RNA half-life depends on at least three components:
1) The enzymes which degrade mRNA;
2) Cis determinants located anywhere in the mRNA but usually in the 3xe2x80x2 terminus of the message; and
3) Trans-acting factors which interact with the message to promote or prevent decay.
There are probably a small number of eukaryotic ribonucleases (20). It is likely that regulated message stability depends on the interaction of binding proteins with specific RNA sequences to either promote or prevent access to ribonucleases.
Fatty acid synthase (FAS) is the cytosolic enzyme that synthesizes palmitate from acetyl-CoA, malonyl-CoA, and NADPH (36). It is rate-limiting in the long-term control of fatty acid synthesis (35) and regulated by several different classes of nutrients (8). FAS overexpression occurs in animal models of obesity (16). Carbohydrates stimulate FAS expression and produce hypertriglyceridemia in animals and humans (35,15).
FAS is also relevant to malignancy. Cultured carcinoma cells maintain high levels of endogenous fatty acid synthesis even in the presence of high concentrations of exogenous fatty acids (24). FAS overexpression in breast and prostate carcinomas is a powerful predictor of a poor clinical outcome (1,9). The enzyme is overexpressed in colorectal tumors (27). Glucose transport and catabolism are enhanced in cancer cells (28) suggesting that coordinate regulation of glucose and lipid metabolism characterize the malignant state.
Glucose alone, independent of hormones, stabilizes the FAS message in HepG2 cells (32). In this model system, glucose maintains message levels for several hours while decay is rapid following glucose deprivation (31).
In accordance with the invention, it has been found that glucose induces the binding of a novel 178 kDa protein to the fatty acid synthase (FAS) message. This glucose-inducible human FAS mRNA binding protein has been purified to homogeneity and its binding element defined. This large phosphoprotein binds to a novel repetitive element in the 3xe2x80x2 untranslated region (UTR) of the FAS mRNA. In particular, the binding has been mapped to a 37 nucleotide stretch within the first 65 bases of the 3xe2x80x2 UTR of mRNA.
Purification and characterization of the novel 178 kDa protein of the invention herein was carried out in conjunction with a determination of the underlying mechanisms whereby glucose stabilizes the mRNA for FAS and its relationship to diverse human disorders, including hyperlipidemia, obesity and malignancy.
More specifically, as described hereinbelow in greater detail:
RNA gel mobility shift assays were used to demonstrate that human HepG2 cells and bovine liver contain a cytoplasmic factor which binds specifically to the 3xe2x80x2 terminus of the human FAS mRNA.
D-glucose increased RNA binding activity by 2.02-fold (p=0.0033) with activity peaking three hours after glucose feeding.
Boiling or treatment of extracts with proteinase K abolished binding.
UV crosslinking of the FAS mRNA-binding factor followed by SDS-PAGE resolved a proteinase K-sensitive band with an apparent molecular mass of 178xc2x17 kD.
The protein was purified to homogeneity using nondenaturing polyacrylamide gels as an affinity matrix.
Acid phosphatase treatment of the protein prevented binding to the FAS mRNA, but binding activity was unaffected by modification of sulfhydryl groups and was not Mg++-or Ca++-dependent.
Deletion and RNase T1 mapping localized the binding site of the protein to 37 nucleotides characterized by the repetitive motif ACCCC and found within the first 65 bases of the 3xe2x80x2 UTR.
Hybridization of the FAS transcript with an oligonucleotide antisense to this sequence abolished binding.
These findings indicate that a 178 kD glucose-inducible phosphoprotein binds to an (ACCCC)n-containing sequence in the 3xe2x80x2 UTR of the FAS mRNA within the same time frame that glucose stabilizes the FAS message. This protein is thus believed to participate in the post-transcriptional control of FAS gene expression.
While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention, it is believed that the invention will be better understood from the following preferred embodiments of the invention taken in conjunction with the accompanying drawings.